Malnutrition kills millions of children worldwide every year. Much of their morbidity and mortality is a result of infections, including those caused by enteropathogens. Efforts to stop these infections have been limited in some populations by unexplained vaccine failures. I hypothesize that there is an interrelationship between malnutrition, the gut microbiome, and the immune system that is causally linked with vaccine responsiveness and that these interrelationships can be informatively probed using 'humanized'gnotobiotic mice. Our lab is conducting longitudinal studies of the gut microbiomes of twins aged 1-36 months living in Malawi who are healthy, or who are concordant or discordant for severe forms of malnutrition (e.g., kwashiorkor). We are addressing the following questions: Are there identifiable configurations of the gut microbiome associated with severe forms of malnutrition? How is the microbiota/microbiome reconfigured with a Ready to Use Therapeutic Food (RUTF) intervention and does it persist after cessation of RUTF? If the configured state does not persist after withdrawal of RUTF, does this mean that in the absence of longer-term nutritional support, lingering abnormalities in the metabolic activities contribute to persistent physiological abnormalities in the human host? We have recently shown that it is possible to efficiently transplant the microbial community present in frozen human fecal samples into germ-free (GF) mice and to transmit the engrafted microbiota from mothers to their offspring. With this capability, I propose two aims for this NRSA application. Aim 1. Use gnotobiotic mice, colonized at 6 weeks of age with de-identified fecal microbiota already collected from several sets of monozygotic twins discordant for kwashiorkor (1 healthy/ 1 malnourished) as well as their mothers'microbiota. Recipients will be fed a macro- and micronutrient deficient Malawi diet, or a control nutritionally 'sufficient'diet. I will follow their responses to mucosal vaccination with cholera toxin and ovalbumin (a model mucosal adjuvant/antigen) using antigen-specific ELISA. I will define their gut mucosal barrier function by serum LPS assays, lactulose/mannitol absorption assays, histo- and immunohistochemical assays of gut morphology and immune cell populations (exploring whether they have features of tropical enteropathy), plus functional genomic studies of gut gene expression. I will perform time series metagenomic studies on their fecal microbiomes (sequencing bacterial 16S rRNA genes, total community DNA, and expressed mRNAs). Aim 2. Assess the role of maternal nutritional and microbiota 'status'on the vaccine responses of their offspring by colonizing pregnant female GF mice with fecal samples from twins discordant for kwashiorkor and their mothers. Mothers will be maintained on either a Malawi or nutrient- sufficient control diet prior to, during and after pregnancy;their offspring will be weaned onto nutrient- deficient or -sufficient diets and subjected to the same vaccination protocols and analyses as described in Aim 1. These experiments should help identify biomarkers of malnutrition and vaccine responsiveness. PUBLIC HEALTH RELEVANCE: Tragically, malnutrition is the leading cause of childhood death worldwide. To understand why malnourished children fail to respond to potentially life-saving vaccines, we are using gnotobiotic mice, colonized with the gut microbial communities harvested from monozygotic twins living in Malawi who are discordant for severe malnutrition, in order to characterize responses to oral vaccination as a function of diet and gut microbial community structure, with the expectation that translation of information gleaned from this mouse model will provide new mechanistic insights and therapeutic strategies for enhancing vaccine responses in vulnerable pediatric populations.